Journal: bioRxiv

Article Title: Systems serology of responses against tumor antigens in ovarian cancer reveal disrupted Fc-mediated immunity

doi: 10.64898/2026.04.25.720834

Figure Lengend Snippet: A) Quantification of Cetuximab, an IgG1 Ab, binding to OVCAR3 cells as compared to patient TBAs by fluorescence. Cetuximab was incorporated at concentrations of 0.01–0.1 µg/mL. B) Quantification of Cetuximab binding to FcγRIIIa as compared to patient TBAs via fluorescence. C) Percent cytotoxicity of unmodified patient TBAs, quantified by an in vitro effector response assay (n=36). All patient TBAs were normalized to the same concentration. Cetuximab was used as a positive control (+). NK cells co-incubated with OVCAR3 cells alone functioned as the negative control (-). A purple bar indicates a patient that was evaluated in A) and B). Data are represented as average ± standard deviation. See also Figure S4.

Article Snippet: OVCAR3 cells (ATCC HTB-161) were grown in complete growth media.

Techniques: Binding Assay, Fluorescence, In Vitro, Concentration Assay, Positive Control, Incubation, Negative Control, Standard Deviation

Journal: Cell Death Discovery

Article Title: A dual role for Caspase8 and NF- κ B interactions in regulating apoptosis and necroptosis of ovarian cancer, with correlation to patient survival

doi: 10.1038/cddiscovery.2015.53

Figure Lengend Snippet: Caspase8 inhibition compounds cytotoxicity in ovarian cancer cells treated with IKK β inhibitor. Caspase8 shRNA toxicity in a sensitization library screen is shown as ( a ) the log2 ratio of untreated versus IKK β -inhibited, or ( b ) log 10 P -value, after 10 days. Caspase8 shRNAs are highlighted in black. ( c ) Cell viability of Ovcar3, Caov3 and PEO1 cells transduced with control shRNA or either of two different Caspase8 shRNAs was measured by XTT after 7 days of exposure to increasing concentrations of IKK β inhibitor. Data are shown as fold control shRNA, in the absence of IKK β inhibitor (DMSO), ±S.E.M., n =8. Asterisks indicate significant differences ( P <0.01) at the concentration range used in subsequent studies. ( d ) Ovcar3 cells were transduced with Caspase8 shRNAs #1, #2, #3 or #4 or control shRNA and exposed to 2.5 μ M IKK β inhibitor or vehicle for 7 days. Viability was measured by XTT and is shown as fold control shRNA and drug control (DMSO). Error bars represent S.E.M., n =8; *P <0.001. ( e ) Ovcar3 cells expressing control or Caspase8 shRNA #2 were treated with 2.5 μ M IKK β inhibitor for 7 days. ( f ) Three ovarian cell lines sensitive to IKK β inhibition (Ovcar3, Caov3 and Igrov1) and one insensitive cell line (Ovcar8) were stained by IHC with NF- κ B-p65 and Caspase8 antibodies and the presence of nuclear protein analyzed. IHC scores for relative amounts of nuclear NF- κ B-p65 and Caspase8 were measured in four to six uniform fields per sample and averaged. Numbers are average scores ±S.E.M.

Article Snippet: Ovcar3 cells were transduced with a lentiviral vector containing an NF- κ B transcriptional regulatory element, using the Cignal Lenti Reporter System (CLS-013L, Qiagen, Valencia, CA, USA).

Techniques: Inhibition, shRNA, Transduction, Control, Concentration Assay, Expressing, Staining

Journal: Cell Death Discovery

Article Title: A dual role for Caspase8 and NF- κ B interactions in regulating apoptosis and necroptosis of ovarian cancer, with correlation to patient survival

doi: 10.1038/cddiscovery.2015.53

Figure Lengend Snippet: IKK β inhibition downregulates NF- κ B signaling and caspase activation after TNF α stimulation. ( a ) Ovcar3 cells expressing control or Caspase8 shRNA were transduced with NF- κ B luciferase reporter. NF- κ B signaling in was measured after 18 h treatment with TNF α (10 ng/ml) and/or IKK β inhibitor (2.5 μ M). Data are arbitrary luciferase units normalized to XTT and are represented as mean±S.E.M.; * P <0.05 based on t -test. ( b ) mRNA expression of Caspase8 and NF- κ B target genes cFLIP, CLDN1, CXCL1, CXCL2 and IL-8 was measured by quantitative RT-PCR in Ovcar3 cells, expressing either control or Caspase8 shRNA. Expression was normalized to that of GAPDH, and expressed as mean±S.E.M. of triplicate experiments. * P <0.05 based on t -test.

Article Snippet: Ovcar3 cells were transduced with a lentiviral vector containing an NF- κ B transcriptional regulatory element, using the Cignal Lenti Reporter System (CLS-013L, Qiagen, Valencia, CA, USA).

Techniques: Inhibition, Activation Assay, Expressing, Control, shRNA, Transduction, Luciferase, Quantitative RT-PCR

Journal: Cell Death Discovery

Article Title: A dual role for Caspase8 and NF- κ B interactions in regulating apoptosis and necroptosis of ovarian cancer, with correlation to patient survival

doi: 10.1038/cddiscovery.2015.53

Figure Lengend Snippet: Necroptosis, but not apoptosis, is prominent in Caspase8-depleted cells. ( a ) Cells expressing either control or Caspase8 shRNA were exposed to Caspase8 inhibitor ZIETD (25 μ M), staurosporine (1 μ M), TNF α (10 ng/ml) and/or IKK β inhibitor (2.5 μ M), to induce caspase activity. Results are expressed as average luciferase units ±S.E.M., n =16. * P <0.05 based on t -test. ( b ) CASPASE 3 and 7 activity was measured in Ovcar3 cells exposed to similar conditions as in a . ( c ) Control shRNA-transduced Ovcar3 cells were exposed to IKK β inhibitor with or without TNF α stimulation, in the presence of apoptosis inhibitor ZIETD, or NEC1, a known inhibitor of necroptosis. Cells were treated for 18 h with TNF α (10 ng/ml), IKK β inhibitor (2.5 μ M), SMAC-mimetic (birinapant 200 nM), and/or ZIETD (25 μ M) or NEC1 (25 μ M) as indicated. Viability was assessed by XTT assay. Results are expressed as average±S.E.M., n =8. ( d ) Caspase8-depleted cells were treated as in b .

Article Snippet: Ovcar3 cells were transduced with a lentiviral vector containing an NF- κ B transcriptional regulatory element, using the Cignal Lenti Reporter System (CLS-013L, Qiagen, Valencia, CA, USA).

Techniques: Expressing, Control, shRNA, Activity Assay, Luciferase, XTT Assay

Journal: Cell Death Discovery

Article Title: A dual role for Caspase8 and NF- κ B interactions in regulating apoptosis and necroptosis of ovarian cancer, with correlation to patient survival

doi: 10.1038/cddiscovery.2015.53

Figure Lengend Snippet: Caspase8-depleted ovarian cancer cells show evidence of cell death by necroptosis. ( a ) Western analysis was performed on cell lysates obtained from Ovcar3 and Caov3 cells expressing control or Caspase8 shRNA #2, after treatment with IKK β inhibitor (2.5 μ M) and/or TNF α (10 ng/ml) for 18 h. Protein levels of Caspase8, RIPK1 (uncleaved, 78 kDa), MLKL and cleaved PARP are shown. GAPDH was used as loading control ( b ) Western analysis was performed on cell lysates obtained from Ovcar3 cells expressing control or Caspase8 shRNA #2, after treatment with IKK β inhibitor (2.5 μ M), SMAC-mimetic (birinapant 200 nM) and/or TNF α (10 ng/ml) for 18 h. Protein levels of Caspase8, RIPK1 (uncleaved, 78 kDa), MLKL, cIAP1 and cleaved PARP are shown (upper). β -Tubulin was used as loading control. ( c ) Ovcar3 cells expressing either control or Caspase8 shRNA #2 were transiently transduced with control or RIPK1 siRNA. ( d ) Ovcar3 cells described in c were exposed to IKK β inhibitor with or without TNF α stimulation, in the presence of apoptosis inhibitor (ZIETD) or necroptosis inhibitor (NEC1). Cells were treated for 18 h with TNF α (10 ng/ml), IKK β inhibitor (2.5 μ M), SMAC-mimetic (birinapant 200 nM) and/or ZIETD (25 μ M) or NEC1 (25 μ M) as indicated. Viability was assessed by XTT assay. Results are expressed as average±S.E.M., n =8, * P <0.01 based on t -test, and shown as percent change from no treatment control.

Article Snippet: Ovcar3 cells were transduced with a lentiviral vector containing an NF- κ B transcriptional regulatory element, using the Cignal Lenti Reporter System (CLS-013L, Qiagen, Valencia, CA, USA).

Techniques: Western Blot, Expressing, Control, shRNA, Transduction, XTT Assay